Genome-wide association studies (GWAS) in rheumatoid arthritis (RA) have led to the identification of more than 25 regions of the genome that contribute to risk of disease (RA risk loci). Despite this exciting progress at gene mapping, these alleles explain <20% of the overall variation in RA risk, indicating that many more risk alleles remain to be identified. In the current application, we propose to re-sequence the protein-coding portion of candidate genes from 25 RA risk loci to search for rare functional mutations. If a rare disease-associated mutation is identified, then this gene is almost certainly the causal gene. These rare functional mutations will provide an opportunity to study fundamental mechanisms of RA pathogenesis. In this application, we hypothesize that rare functional alleles contribute to risk of RA in genes that also harbor independent common alleles identified by GWAS. To test this hypothesis, we will implement next-generation sequencing technology, which provides a cost-effective approach to re- sequence candidate genes. Aim 1: Re-sequence the coding exons of 27 biological candidate genes in 750 RA cases and 750 matched controls using next-generation sequencing technology. We will identify all common and rare alleles using a robust sequencing pipeline recently developed by our colleagues at the Broad Institute. Aim 2: Genotype all functional alleles in the same 750 cases and 750 controls, and conduct an association study with disease risk. We will genotype all putative functional mutations in the same samples to confirm the sequence calls and to determine the genotype of each subject. We will test for association with RA risk in two ways: an allele-based test and a gene-base test. Aim 3: We will replicate our findings in additional samples. If our allele-based test identifies disease-associated functional alleles that are low frequency yet segregate on an ancestral haplotype, then we will replicate the finding by genotyping the rare allele in >10,000 additional case-control samples. If our gene-based test identifies a collection of disease-associated rare alleles that are private to individuals (i.e., are of recent ancestral origin, do not segregate on ancestral haplotypes), then we will re-sequence the coding exons of that gene in an additional 1,500 cases and 1,500 controls. We have assembled a team of scientists uniquely positioned to conduct all aspects of the project. Our studies will be conducted within a scientific environment that appreciates the power of next-generation sequencing, without underestimating it's technical and analytical challenges. PUBLIC HEALTH RELEVANCE: A long-term goal of understanding the genetic basis of rheumatoid arthritis is to improve care of patients with this common and debilitating disease. In theory, identifying specific pieces of DNA ("alleles") should aid in diagnosing a treatable condition either prior to onset of symptoms, or early in the course of disease before bone destruction occurs. In addition, genetics should provide insight into important steps of the disease pathway, allowing for the development of new therapies that target these pathways in at-risk individuals.